Requirement for Pdx1 in specification of latent endocrine progenitors in zebrafish

<p>Abstract</p> <p>Background</p> <p>Insulin-producing beta cells emerge during pancreas development in two sequential waves. Recently described later-forming beta cells in zebrafish show high similarity to second wave mammalian beta cells in developmental capacity. Loss-of-function studies in mouse and zebrafish demonstrated that the homeobox transcription factors Pdx1 and Hb9 are both critical for pancreas and beta cell development and discrete stage-specific requirements for these genes have been uncovered. Previously, exocrine and endocrine cell recovery was shown to follow loss of <it>pdx1 </it>in zebrafish, but the progenitor cells and molecular mechanisms responsible have not been clearly defined. In addition, interactions of <it>pdx1 </it>and <it>hb9 </it>in beta cell formation have not been addressed.</p> <p>Results</p> <p>To learn more about endocrine progenitor specification, we examined beta cell formation following morpholino-mediated depletion of <it>pdx1 </it>and <it>hb9</it>. We find that after early beta cell reduction, recovery occurs following loss of either <it>pdx1 </it>or <it>hb9 </it>function. Unexpectedly, simultaneous knockdown of both <it>hb9 </it>and <it>pdx1 </it>leads to virtually complete and persistent beta cell deficiency. We used a <it>NeuroD:EGFP </it>transgenic line to examine endocrine cell behavior <it>in vivo </it>and developed a novel live-imaging technique to document emergence and migration of late-forming endocrine precursors in real time. Our data show that Notch-responsive progenitors for late-arising endocrine cells are predominantly post mitotic and depend on <it>pdx1</it>. By contrast, early-arising endocrine cells are specified and differentiate independent of <it>pdx1</it>.</p> <p>Conclusions</p> <p>The nearly complete beta cell deficiency after combined loss of <it>hb9 </it>and <it>pdx1 </it>suggests functional cooperation, which we clarify as distinct roles in early and late endocrine cell formation. A novel imaging approach permitted visualization of the emergence of late endocrine cells within developing embryos for the first time. We demonstrate a <it>pdx1</it>-dependent progenitor population essential for the formation of duct-associated, second wave endocrine cells. We further reveal an unexpectedly low mitotic activity in these progenitor cells, indicating that they are set aside early in development.</p